Cardiovascular diseases, which result from thrombosis of critically situated blood vessels, are a leading cause of deaths. At present, the standard treatment is by dissolution of the thrombus using a thrombolytic agent, namely a plasminogen activator (PA). Activation of plasminogen by the PA agent produces plasmin, which then degrades fibrin. Plasmin, however, also degrades other clotting factors. Thrombolytic therapy, which introduces systemic generation of plasmin, therefore carries the risk of bleeding. Previously, we proposed a novel, pro-drug and triggered-release approach (termed Antibody _Targeted_Triggered _Electrically Modified _Prodrug._Type_Strategy;AT-rEMPTS.) which could permit targeted thrombolysis without the risk of bleeding. The approach consists of 2 components: [i] a fibrin-targeting antibody linked to an anionic heparin (termed Ab-Hep); and [ii] a cation-modified PA (termed m-PA+). These 2 components are linked automatically via an electrostatic interaction. Since the cations used for PA modification are small, m-PA+ would retain its fibrinolytic activity. This activity, however, would be inhibited after binding to Ab-Hep, due to blockage of the PA's catalytic site by the appended macromolecules. Since protamine is a clinical heparin antagonist with an unmatched heparin-binding affinity, it can be used safely and effectively to trigger the release of m-PA+ from the Ab-Hep/m-PA+ complex at site of the thrombus. Therefore, the approach would permit administration of a strong fibrin-targeting but inactive PA drug, and subsequently a triggered release of the active PA drug in close proximity of the fibrin deposit. Such features would not only enhance the potency and fibrin-selectivity of the PA drug, but also attenuate the bleeding risk by permitting the drug to specifically attack the concerned components of a thrombus while sparing other circulating factors. Remarkable progress and outstanding productivity have been achieved during the previous grant period. As a consequence, 30 peer-reviewed manuscripts and 17 abstracts have been published or submitted within a period of less than 4 years. All the specific aims proposed in the previous application have been successfully accomplished. Most importantly, progress made and pitfalls identified during the previous grant period led to the establishment of a fine-tuned research plan and strategy, including the use of: (i) reversed ATTEMPTS (termed re-ATTEMPTS) strategy;(ii) computer simulation technology to identify the ideal site in t-PA for peptide modification;(iii) point-mutation methodology to achieve site-specific incorporation of peptide to t-PA; and (iv) modern physiologically-based pharmacokinetics(PBPK) approach to optimize the dosing time of the triggering agent to achieve the utmost goal of effective and safe thrombolytic therapy. The 4 broadly integrated Specific Aims are: Aim [i]: syntheses of an anti-fibrin antibody-linked, modified-t-PA complex (termed Ab/mt-PA)consisting of a poly(Glu)-modified anionic t-PA (termed m-PA) and a LMWP-modified cationic antibody (termed LMWP-Ab); Aim [ii]: in vitro characterization of the Ab/mt-PA complex using a flow-type plasma clot assay; Aim [iii]: in vivo investigation of the targeting pharmacokinetics for assessing the optimal dosing time for the triggering agent heparin; and Aim [iv]: in vivo evaluation of the efficacy and safety of the re-ATTEMPTS system using both the rat IVC thrombosis model and the canine intra-cornonary thrombosis model.